3, 11-dioxygenated 17alpha-carboxyethylandrost-4-en-17beta-ol lactone, nor compoundscorresponding, and manufacturing processes



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United States Patent 0 3,043,837 3,11 DIOXYGENATED 17 a CARBOXYETHYLAN- DROST-4-EN 17B 0L LACTONE, NOR COM- POUNDS CORRESPONDING, AND MANUFAC- TURING PROCESSES John A. Celia, Skokie, 111., assighor to G. D. Searle & C0., Chicago, [1]., a corporation of Delaware No Drawing. Filed Aug. 17, 1959, Ser. No. 833,956 11 Claims. (Cl. 260-23957) wherein R represents hydrogen or a methyl radical; A

represents an alkylene or alkenylene radical, especially Equivalent to the foregoing lactones for purposes of the present invention are the corresponding hydroxy acids and their alkali salts, of the formula wherein R, A, X, and Y have the meanings assigned before, there being a 4(5), 5(6), or 5(10) double bond present in each instance; and M represents hydrogen, an alkali metal, or the ammonium radical. Those skilled in the art will appreciate that the described salts are readily derived from the corresponding lactones on contact with aqueous alkali. The free acids, in turn, are obtained from the salts by a critically brief exposure to a proton source; prolongation of the exposure time induces lactonization.

The application for Letters Patent securing the invention herein described and claimed is a continuation-in-part of applicants prior copending application, Serial No. 682,626, filed September 9, 1957, and now abandoned.

The compounds of this invention are useful because of their valuable pharmacological properties. Thus, for example, the subject compositions are diuretic agents wherein ll-oxygenation serves to surprisingly enhance the ratio of oral to parenteral activity.

Manufacture of the compounds herein disclosed and 3,943,837 Patented July -10, 1962 "ice claimed proceeds from ll-methylenic lactones of the formula John A. Cella in United States Patent No. 2,705,712 and his application for United States Patent No. 741,473, filed June 12, 1958. -These lactones, although apparently impervious to ll-oxygenation by such well-known hydroxylaters as Aspergillus and Penicillium, upon fermentation with a culture of Rhizopus are converted to the corresponding Ila-'COIIIPOHDdS hereof, and on perfusion through excised, surviving, desirably lacerated, mammalian adrenal glands afford the apposite llB-compounds. The ll-hydroxy compounds, in'turn, are further oxidized to corresponding ll-ketones on contact with chromic anhydride in'aqueous acid acetonic medium.

The fermentation procedure is carried out in a suitably nutritive medium preliminarily inoculated with .spores of Rhizopus fungus and maintained at about 25 with agitation under aerobic conditions conductive to the development of submerged growth. The steroid to be oxygenated is preferably added to the flourishing culture in solution ,for example, in acetone or propylene glycol whereupon the fermentation is continued as before until'such time as the oxidation is substantially complete-representatively, after upwards of 24 hours. Thefermentation medium is then extracted with an appropriate solvent, such as d-ichloromethane, and the desired product obtained as the residue following vacuum distillation.

In the perfusion process, glands from such ascattle, horses, pigs, rabbits, and other common animals are used, those from larger animals beingmore convenient, and cow adrenals.being-especially preferred. The glands are carefully dissected from the anesthetized or freshly killed animal following laparotomy and quickly transferred to coldaqueous saline for prompt perfusion. Extraneous fat and tissue are removed from each gland, and small venal entries near the main large vena cava are tied off. A suture is looped around the neck of the adrenal vein, and a cannula is pressed in and tied. Preferably, multiple cortical laceration over substantially the entire surface of the gland'is effected, the lacerations being spaced from 0.5 to 3, millimeters apart. through a'depth of A to /2 the thickness of the cortical layer, and care being exercised to avoid mutil-ating the deeper medular layer of the gland. Aqueous saline is applied to the cannula under a pressure of 20-50 millimeters of mercury to free the system from air, flush it, and insure an adequate flow rate. During this operation, all fat unembianous tissue solution aifords a particularly efficacious perfusion medium. The tendency of blood-containing media to clot can be minimized by preliminary perfusion through liver.

Per-fusions are ordinarily carried out at temperatures a in the range, 3540 'centigrade, the perfusion media being supplied at pressures of the order of 20-100 millimeters of mercury by means of circulating pumps. One or a plurality of (parallel) glands is employed, and the time of perfusion varies from a few minutes to several hours, depending upon the rate of flow and the volume of material to be perfused. A flow rate of 3 to 6 liters per gland per hour is generally satisfactory both as to conversion of starting material and purity of product. At least one pass through the glands(s) is, of course, indicated; and additional cycles may increase the yield of oxygenated product where input concentrations of material to be hydroxylated are comparatively high. The preferred concentration of starting material is 50-300 milligrams per liter, but as little as 50 and as much as 1000 milligrams per liter may be perfused on occasion. Incorporation of the starting material is facilitated by first dissolving it in a small amount of solvent, for example, propylene glycol.

It is essential that perfusion be commenced as rapidly as possible after preparation of the gland is completed, and the entire procedure is best performed as aseptically as conditions will permit. Practical sterility can be maintained by addition to the medium of suitable concentrations of antibiotics such as penicillin and streptomycin. Throughout the course of the perfusion, oxygen is continuously fed into the perfusion liquid to insure maximum survival of the gland(s) The 11 fl-hydroxylated lactones present in the perfusate can be isolated by a variety of procedures known in the art, perhaps the simplest of which involves merely extracting with a solvent-tor example, isopropyl acetate which is then stripped by evaporation under reduced pressure, leaving the desired product as a residue which can be further purified by recrystallization from benzene or the equivalent. As an alternative to perfusing the corresponding ll-met-hylenic material in deriving the 3-hydroxy-S-enic lacetones of this invention, the latter materials can be prepared from corresponding 3-oxo-4-enes wherein the ll-oxygen function has been introduced, by '3-enolization with isopropenyl acetate followed bysodium .borohydride reduction in alcohol.

The following examples describe in detail compounds illustrative of the present invention and methods which have been devised for their manufacture. However, the invention is not be be construed as limited thereby, either in spirit or in scope, since it will be apparent to those skilled in the art of organic synthesis that many modifications, both of materials and of methods, may be practiced without departing from the purpose and intent of this disclosure. Throughout the examples hereinafter set forth, temperatures are given in degrees centigrade, pressures in pounds per square inch (p.s.i.) or millimeters (mm.) of mercury, and relative amountsof materials in parts by weight, except as otherwise noted.

Example 1 "fermentation tank is charged with a nutrient medium containing, per 1000 parts of tap water, 33 parts of dextrose, parts of commercial cotton seed meal flour, 3 parts of corn steep liquor, and 2 parts of silicone antifoam emulsion Tank and medium are sterilized by heating to temperatures in the range, 110120, and then cooled to about 25 whereupon the medium is inoculated with an aqueous suspension of spores from a culture of Rhizopus sp. A.T.C.C. 13429. The medium is main- 5 tained at about 25 for 29 hours, during which time a stream of sterile air is passed through and the developing culture is agitated to produce submerged growth. Suflicient 170:. (2 -carboxyethyl) 17,8 hydroxyandrost- 4-en-3-one lactone dissolved in a minimal quantity of acetone is then introduced to bring the concentration of steroid to one part per 3000 parts of medium. Agitation with aeration at about 25 is thereupon resumed for 12 hours, at the end of which time the resultant mixture is extracted with dichloromethane. -The extract is dried over anhydrous sodium sulfate and stripped of solvent by distillation. The residual oil, on trituration with anhydrous ether, crystallizes. Recrystallization from methanol affords the desired l7u-(2-carboxyethyD-1la,17fi-dihydroxyandrost 4 en 3 one lactone hemimethanolate melting at 173-174. The product is further characterized by a maximum in the ultraviolet spectrum at 241 millimicrons with a molar extinction coefiicient of 15,900. The specific rotation of a dioxane solution, referred to sodium D, is +48. The product has the formula 25 Example 2 17a (2 -carb0xyethyl) 415,17fl-dihydroxyandr0st-4- en-3-0ne -lact0ne.A solution of one part of 17u-(2- carboxyethyl)-l7p-hydroxyandrost-4-en-3-one lactone in 40 parts of propylene glycol is added to 250 volumes of a perfusion medium made by mixing 7 parts of citrated whole cow blood with 5 parts of modified (calcium chloride omitted) Tyrode solution and subjecting the resultant mixture of the action of a stream of oxygen bubbling therethrough over 2 /2 hours period. Perfusion at 3637.5 C. through 8 cow adrenals averaging 17.9 grams each is straightway commenced, the glands in question having been preliminarily trimmed, cannulated, and otherwise prepared in accordance with the technique hereinbefore described. During the course of the perfusion, oxygen is continuously bubbled through the medium. After approximately 3 hours, in the course of which 6 passes of the medium through the glands are completed, perfusion is stopped; and the perfusate is thrice extracted with isopropyl acetate. Solvent is removed from the combined extracts by evaporation under reduced pressure, and the residue is crystallized from ethyl acetate. The product thus obtained is l7a-(2-carboxyethyl)-l1,8, l7p1-dihydroxyandrost-4-en-3-one lactone, which melts at 203-207" C. and has the formula a i H O\ Example 3 1 7a (Z-carboxyethyl) -175-h drxyandrost-4-ene-3,I 1- dione lact0ne.To a solution of 6 partsof 17u-(2-carboxyethyl) l15,17,8-dhydroxyandrost-4-en-3-one lactone in 400 parts of acetone at room temperatures is added, with vigorous agitation, a mixture of 2 parts of chromic anhydride, 3 parts of concentrated sulfuric acid, and parts of water. Excess oxidant is destroyed with 2-propanol, whereupon the resultant mixture is filtered and the filtrate evaporated to dryness in vacuo. The residue, recrystallized from anhydrous ethanol, is 17u-(2-carboxyethyl)-17B-hydroxyandrost-4-ene-3,1l-dione lactone melting at 255258 C. The product has the formula HaC Example 4 17a. (2 carboxyethyl) 11 u,] 7p-dihydroxy-19-riorana'r0st-4-en-3-one y-lactona-A stainless steel fermentation tank is charged with a nutrient medium containing, per

I dichloromethane.

1000 parts of tap water, 27 part of dextrose, 5 parts of commercial cotton seed meal flour, 3 parts of corn steep liquor, and 2 parts of silicone anti-foam emulsion. Tank and medium are sterilized by heating to temperatures of the order of 110-120 and then cooled to about 25, whereupon the medium is inoculated with an aqueous suspension of spores from a culture of Rhizopus sp. A.T.C.C. 13429. The medium is maintained at about 25 for 48 hours, during which time a stream of sterile air is passed through and the developing culture is agitated to produce submerged growth. Sufiicient 17oc-(2- carboxyethyl)-l7fi-hydroxy-19-norandrost-4-en-3-one lactone dissolved in a minimal quantity of acetone is then introduced to bring the concentration of steroid to 1 part per 3000 parts of medium. Agitation with aeration at about 25 is thereupon resumed for 16 hours, at the end of which time the resultant mixture is extracted with The extract is dried over anhydrous sodium sulfate and stripped of 'solventby distillation. The residual oil, on trituration with anhydrous ether, crystallizes. Recrystallization from ethyl acetate affords the desired 17a-(2-carboxyethyD-1lu,l7;3-dihydroxy-l9- norandrost-4-en-3-one 'y-lactone melting at 140-142". The product is further characterized by a maximum in the ultaviolet spectrum at 240.5 millimicrons, with a molar extinction coefficient at 17,000. The product has the formula Example 5 17a (2 carboxyethyl) 11,3,1 2'fl-dihydroxy- 19-noran- V 6 ample 1, 1 part of 17a-(2-carboxyethyl)-l7fi-hydroxy-19- norandrost-4-en-3-one lactone is perfused through 8 cowv adrendals averaging 17.8 grams each to give, after 3 hours during which 11 passes occur, l7a-(2-carboxyethyl)-11B, 17fl-dihydroxy-19-norandrost-4-en-3-one lactone, which is purified by consecutive recrystallization from benzene and anhydrous ethanol. The product thus obtained melts at 213-215" C., solidifies above this melting point and melts again at 220-222 C., and has the formula Example 6 What is claimed is: 1. A compound of the formula Y RI wherein R is selected from the group consisting of hydrogen and methyl radicals and Y is selected from the group consisting of hydroxymethylene and carbonyl radicals.

2. 17a (2-carboxyethyl)-11a,l7B-dihydroxyandrost-4- en-3-one -lactone hemimethanolate.

4. 17oz (Z-carboxyethyl)-17/3-hydroxyandrost-4-ene-3, ll-dione lactone.

5. 17a (2-carboxyethyl)-l1a,17,8-dihydroxy-19-norandrost-4-en-3-one 'y-lactone.

6. 17a (Z-carboxyethyD-l1 8,l7fi-dihydroxy-l9-norandrost-4-en-3 one 'y-lactone.

10. In a process for the manufacture of compounds of the formula 7. 17oz (2 carboxyethyl)l7fi-hydroxy-19-norandrost- 4-ene 3,1 1-dione 'y-lactone.

8. In a process for the manufacture of compoundsof the formula O a T O Y 10 i 3 R O:

wherein R is selected from the group consisting of hydro- O: gen and methyl radicals and Y is selected from the group consisting of fl-hydroxymethylene and carbonyl radicals, the step which comprises perfusing an excised, surviving, wherein R is selected from the group consisting of hydrobeef adrenal gland with an ll-methylenic steroidal lactone gen and methyl radicals and Y is selected from the group' 2 of the formula consisting of u-hydroxymethylene and carbonyl radicals, 0 O the step which comprises contacting with enzymes from F a culture of Rhizopus A.T.C.C. 13429 and ll-methylenic 0 steroidal lactone of the formula 0 F R l o HaC\ R R being defined as before.

11. In a process for the manufacture of compounds of the formula 0: I 0 R bemg defined as before. 9. In a process for the manufacture of compounds of the formula Y R l o O O: s(\ V wherein R' is selected from the group consisting of hydrogen and methyl radicals and Y is selected from the group consisting of B-hydroxymethylene and carbonyl radicals,

r the step which comprises perfusing an excised, surviving,

cortically-lacerated, beef adrenal gland with an ll-methylenic steroidal lactone of the formula V 0 wherein R is selected from the group consisting of hydrogen and methyl radicals and Y is selected from the group consisting of a-hydroxymethylene and carbonyl radicals,

the step which comprises aerobically contacting with a a submerged culture of Rhizopus A.T.C.C. 13429 an 11- R methylenic steroidal lactone of the formula 0 at 3637.5 in a medium derived and maintained by He? bubbling oxygen through a mixture of citrated beef blood and Tyrode solution modified to the extent that calcium chloride is not present, R in the formula for the ll-meth- R ylenic lactone being defined as before.

References Cited in the file of this patent UNITED STATES PATENTS 2,658,022 Haines et a1. Nov. 3, 1953 2,705,712 Oella Apr. 5, 1955 at 25 in a nutrient medium, R in the formula for the 2,844,513 Wettstein et al. July 18, 1956 Cella Dec. 22, 1959 ll-methylenic lactone being defined as before. 2,918,463

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent N0. 3,043 837 July 10, 1962 John A. Cella It is hereby certified that error appears in the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

Column 2, line 30, for "conductive" read conducive column 3, line 49, for "lacetones" read lactones column 4, lines 25 to 38, to the right of the formula for "JCO OH" read JCH OH column 5, lines 60 to 71, the formula should appear as shown below instead of as in the patent:

Signed and sealed this 30th day of July 1963.

(SEAL) Attest:

ERNEST W. SWIDER DAVID L. LADD Attesting Officer Commissioner of Patents 

1. A COMPOUND OF THE FORMULA 